The present invention relates to a medium for use in cultivating microorganisms that does not precipitate in the presence of lead.
Quantitative study of Pb2+ uptake in microorganisms is important for developing technologies to remove and/or stabilize Pb2+ in soils and sediments. There have been several studies concerning Pb2+ uptake (15), the effect of Pb2+ on growth (8) and Zn2+ uptake (13), chemical transformation of Pb2+ by bacteria (2), and Pb2+ associated with cells (1). These studies are conducted to study the interaction of Pb2+ with organisms either as models for human-Pb2+ interactions or for using organisms to remediate lead contaminated sites.
However, a direct and quantitative analysis of Pb2+ by these and other studies has been frustrated by the insolubility of Pb2+ precipitates because a quantitative analysis of Pb2+ absorption or adsorption by bacteria requires that the cells be isolated in a manner which prevents interferences from Pb2+, and Pb2+ precipitates in the external medium when present in concentrations of generally less than 1 mM. If an insoluble Pb2+ precipitate coexists with cells in the medium, the precipitate cannot be separated and removed without destroying the bacteria. Since the Pb2+ precipitate is typically highly insoluble, it cannot be dissolved by simply increasing the volume of the washing solution used in standard centrifugation or filter washing methods.
Another problem is that Pb2+ precipitate prevents accurate turbidity measurements (the quickest measure of cell density) because the precipitate scatters light, creating an artificially high cell density reading.
To avoid or minimize Pb2+ precipitation, some workers have used very low concentrations of phosphate in minimal medium (10, 12, 13), or diluted rich media (e.g., ref. 8). The drawback to using these media is that they are limited to very low Pb2+ concentrations, typically micromolar. If a microorganism accumulates Pb2+ to a cytoplasmic concentration of 1-2 mM, this would readily deplete an extracellular concentration of 1 microM Pb2+. Further, this variation of extracellular Pb2+ concentration significantly complicates a quantitative analysis of Pb2+ uptake kinetic data. Another drawback is that these media usually only support minimal cell densities. The limitation of low cell densities results from the small amount of time in exponential growth which has a high enough cell density, and, hence, a reasonable volume of culture, sufficient to detect quantitatively Pb2+ associated with the bacteria.
Alternatively, to study the effects of Pb2+ on bacteria and the chemical transformation of Pb2+ by bacteria, many workers have used media containing lead precipitate. This precipitate can occur either initially (i.e., LB medium in ref. 8) or can be consequent with growth (e.g., defined citrate medium in reference 2). However, in both cases there is no developed method to quickly separate precipitate from intact cells, and thus these media cannot be used to quantity Pb2+ uptake in bacteria.
It is an object of the present invention to overcome the aforementioned differences in the prior art.
It is another object of the present invention to provide a minimal medium which does not precipitate with concentrations of over 1 mM Pb2+.
It is a further object of the present invention to provide a medium which has a source of phosphate for microorganisms, but which does not precipitate in the presence of Pb2+.
According to the present invention, a minimal medium has been developed which does not precipitate with Pb2+ in concentrations up to 25 mM and which supports growth of soil isolate up to typical minimal medial cell density of xcx9c3xc3x97109 CFU/mL and up to xcx9c8xc3x97109 CFU/mL when the glucose concentration is increased to 200 mM. The medium of the present invention uses a phosphate substitute to provide a source of phosphate for the bacteria which does not precipitate lead.
Of the compounds which can be used, O-phosphate-L-threonine is particularly preferred as a source of phosphate for the microorganisms which do not precipitate with Pb2+.